The Role Of TRB3 In Global Cerebral Ischemia And Reperfusion Injury And Itsmechanism

Objective:To explore the changes of TRB3 in hippocampus induced by global cerebral ischemia and reperfusion injury.To construct and identify TRB3 shRNA lentivirus and tranfected into hippocampus of rats.To explore the effect of TRB3 shRNA interference on the substrates of TRB3 as well as ER stress and neuronal apoptosis after GCI/R.To investigate the role of TRB3 and its mechanism in GCI/RMethods:1.The influence and its mechanism in TRB3 protein expression of hippocampus during global cerebral ischemia and reperfusion injuryTen mins of global cerebral ischemia was performed on 48 adult male SD rats,using the method of 4-vessel occlusion.Rats were sacrificed for decapitation at 1 h,6h 12 h 24 h,72 h of reperfusion.Immunofluorescence staining was used to detect the expression of TRB3 in normal hippocampus of rats.Tunnel assay was used for testing the apoptosis of neurons in response to ischemia.The expression of TRB3 and its substrate(p-Akt),the apoptosis-associated proteins(such as Cleaved Caspase-3,Bax and Bcl-2)and ER stress markers(CHOP,ATF4 and Caspase-12)were measured by western blot.2.Construction of TRB3 shRNA interference lentivirus and transfection.Gene recombinant were constructed by Shanghai Genechem Co.Ltd.18 SD rats were randomly divided into three groups.Control group(Con group),TRB3 shRNA interference lentivirus group(Con+LV-TRB3-RNAi group)and Negative control lentivirus group(Con+LV-control group).TRB3 shRNA interference lentivirus,negative control lentivirus were injected to CA1 region of hippocampus by stereotaxic injection.The expression of GFP was examined by immunostaining 72 h after stereotaxic injection.The expression of TRB3 protein was examined by western blot.3.The impact of TRB3 RNA interference on the apoptosis of neurons induced by GCI/R and its mechanism.24 adult male SD rats were used and divided into four groups:Control group(Con group),ischemia group(I/R 72 h group),TRB3 shRNA lentivirus+ischemia group(LV-TRB3-RNAi+I/R 72 h group),control lentivirus group+ischemia group(LV-control+I/R 72h group).LV-TRB3-RNAi and LV-control were bilaterally injected into the CA1 region of the hippocampus through stereotaxic injection 72 h before GCI/R model was performed.72h after GCI/R was performed,the protein level of TRB3,p-Akt,apoptosis-related proteins(Cleaced Caspase-3,Bax and Bcl-2)and ER stress markers(CHOP,ATF4 and Caspase-12)were measured by western blot.The apoptosis of neurons were detected by TUNEL assay.Results:1.The influence and its mechanism in TRB3 protein expression of hippocampus during global cerebral ischemia and reperfusion injuryTRB3 was expressed in CA1,CA2,CA3 and DG region of hippocampus.TRB3mainly expressed in neurons.GCI/R 72 h enhanced TUNEL-positive cells in the CA1region of hippocampus.Compared with Con group,the expression of TRB3 in hippocampus was significantly increased after GCI/R and the substrate of TRB3(p-Akt)was decreased(P<0.05).Meanwhile,GCI/R induced the up-regulation of Cleaved caspase-3 and Bax expression as well as down-regulation of Bcl-2 protein expression in the hippocampus in I/R group(P<0.05).Furthermore,the protein level of CHOP,ATF4 and Caspasd-12 were increased markedly in response to GCI/R compared with Con group(P<0.05).2.Construction of TRB3 shRNA interference lentivirus and transfection.The tittering of TRB3 shRNA interference lentivirus was 5×10~8 TU/ml.A wide range of GFP expression was detected by immunofluorescence in Con+LV-TRB3-RNAi group and Con+LV-control group.Compared with Con group and Con+LV-control group,TRB3 mRNA and protein level were significantly decreased by TRB3 RNA interference(P<0.05).3.The impact of TRB3 shRNA interference on the apoptosis of neurons induced by GCI/R and its mechanism.Compared with I/R 72 h group,down-regulation of TRB3,CHOP,ATF4,Caspase-12,Bax,Cleaved Caspase-3,Bax and up-regulation of p-Akt and Bcl-2 were observed in LV-TRB3-RNAi+I/R 72 h group(P<0.05).TUNEL-positive cells in LV-TRB3-RNAi+I/R 72 h group were markedly lower than that in I/R 72 h group and LV-control+I/R 72 h group.Conclusions:1.The expression of TRB3 is elevated after GCI/R,which suggests that up-regulation of TRB3 may be involved in ER stress and apoptosis of neurons in response to GCI/R.2.TRB3 shRNA interference lentivirus was successfully constructed,which can decrease the expression of TRB3.3.Down-regulation of TRB3 can protect neurons against apoptosis through Akt pathway and alleviating ER stress.